Method for purifying PrPres from a biological sample and applications

ABSTRACT

The invention concerns a method for purifying PrPres from a biological sample to be used for qualitative and/or quantitative determination of the PrPres in said sample. The method essentially consists in: (1) incubating, during 30 seconds to 2 hours, at a temperature less than 80° C., said biological sample with a buffer solution A comprising at least a surfactant in an amount ranging between a quarter and four times the weight of the biological sample and optionally a protease, to form a suspension S 1 ; (2) adding to said suspension S 1  resulting from (1) a buffer solution B in an amount sufficient for thinning said suspension, which buffer solution B consists of a solvent or mixture of solvents, which does not solubilize the PrPres and has a constant dielectric ranging between 10 and 25; (3) centrifuging the suspension S 2  resulting from step (2); and (4) solubilizing said pellet in a buffer solution C comprising at least a surfactant and/or at least a chaotropic agent, at a temperature ranging between room temperature and 100° C.

The present invention relates to a novel method of purifying PrPres froma biological sample in order to use it for the qualitative and/orquantitative detection of PrPres in said sample.

Transmissible subacute spongiform encephalopathies are caused bynon-conventional transmissible agents (NCTA), also called prions, theprecise nature of which is still unknown at the present time. TSSEcomprise essentially Creutzfeldt-Jakob disease (CID) in humans, scrapiein sheep and goats and bovine spongiform encephalopathy (BSE) in cattle;other encephalopathies have been revealed in mink or certain wildanimals such as stag and elk.

The outcome of these diseases is inevitably fatal and no effectivetreatment is currently available.

In transmissible subacute spongiform encephalopathies, there is anaccumulation of a host protein, PrP (or prion protein), in an abnormalform (PrPres), mainly in the central nervous system; PrPres copurifieswith the infectiousness and its accumulation precedes the appearance ofhistological lesions. In vitro it is toxic to neuron cultures.

Two biochemical properties usually make it possible to distinguishbetween PrPres and normal PrP: PrPres is partially resistant toproteases and is insoluble in anionic surfactants.

To be able to detect the PrPres present in a sample, it is necessary tosubject said sample to different operations in order to enrich it inPrPres, while eliminating the normal PrP, so that the PrPres can then bedetected by any appropriate specific method without causing:

false positives due to the presence of normal PrP or other contaminants,or

false negatives due to an insufficient concentration of PrPres in thefinal biological sample.

A number of methods of isolating and/or purifying PrPres have beenproposed for this purpose. They are essentially based on the methoddeveloped by Hilmert and Diringer (Nature, 1983, 306, 476-478) andgenerally involve an extraction with a detergent, differentialultracentriugations and a treatment with proteolytic enzymes (MulthaupG. et al., EMBO J., 1985, 4, 6, 1495-1501; Takahashi K et al.,Microbiol. Immunol., 1986, 30, 2, 123-131; Hope J. et al., EMBO J.,1986, 5, 10, 2591-2597; Grathwohl K. U. D. et al., Arch. Virol., 1996,141, 1863-1874; Kascsak R. J. et al., Immunol. Investig., 1997, 26,259-268; R. E. Race et al., J. Gen. Virol., 1992, 73, 3319-3323; Doi etal., J. Gen. Virol., 1988, 69, 955-960; T. Muramoto et al., Am. J.Pathol., 1993, 143, 5, 1470-1479; Farquhar C. F. et al., Gen. Virol.,1994, 75, 495-504 and J. Gen. Virol., 1996, 77, 1941-1946). They havethe disadvantage of comprising a large number of steps including severalultracentrifugations, which are cumbersome to carry out and result incumulative losses of PrPres; these in turn lead to an insufficientsensitivity to obtain a high-quality detection threshold andquantification of the PrPres.

These various methods require research laboratory equipment andimplementation times which are incompatible with use in the field,particularly in abattoirs.

Now, there is a need for rapid verification of the absence or presenceof a transmissible subacute spongiform encephalopathy at the time whenthe animal is slaughtered.

Consequently the inventor set out to provide a method of purifying abiological sample in order to use it for a rapid and reliable detectionof PrPres, said method being sufficiently simple to carry out that itcan be used in the field, especially in abattoirs, and thereby meetingpractical needs better than the methods of the prior art. In fact, themethod according to the invention is:

simple to carry out,

reliable and

easy to interpret it increases the detection sensitivity threshold ofPrPres by eliminating the false positives (normal PrP and othercontaminants), and it eliminates the false negatives because it enablesa substantial amount of PrPres to be obtained, in absolute terms, sinceit is possible to treat large amounts of biological material with apurification yield in excess of 80%; this is of particular value inabattoirs and produces samples in which PrPres is readily detectablewith customary diagnostic tests.

The present invention relates to a method of purifying PrPres from abiological sample, characterized in that it comprises essentially:

(1) the incubation, for 30 seconds to 2 hours, preferably for 30 secondsto 10 minutes, at a temperature below 80° C., of said biological samplewith a buffer A comprising at least one surfactant in an amount ofbetween a quarter and four times, preferably of between a quarter andone and a half times, the weight of the biological sample, andoptionally prior, subsequent or simultaneous incubation with a protease,to form an opalescent to turbid micellar or lamellar suspension S1;under the temperature and quantity conditions mentioned above, whateverthe surfactant or surfactant mixture may be, it does not solubilize mostof the PrPres, which remains in suspension, whereas the normal PrP issolubilized, or even destroyed, if protease is added; said incubation ispreferably carried out at a temperature below 50° C., in the presence ofan amount of surfactant of between a quarter and one and a half timesthe weight of the biological sample; according to the invention, theprotease can in fact be added either before, after or simultaneouslywith the surfactant;

(2) the addition, to said micellar or lamellar suspension S1 obtained in(1), of a buffer B in an amount suitable for clarifying said suspension(for example by forming a microemulsion or a microsuspension), saidbuffer B consisting of a solvent or solvent mixture which does notsolubilize the PrPres and has a dielectric constant of between 10 and25; this gives a suspension S2 which is limpid to the naked eye;

(3) the centrifugation of the suspension S2 obtained in step (2); saidcentrifugation is carried out for example for 2 to 10 minutes at a speedbelow 20,000 g, preferably at a speed of between 3500 g and 17,500 g;the PrPres ends up in the centrifugation residue with a PrPrespurification yield surprisingly of between 80 and 100%; advantageouslythe centrifugation time and speed can be adapted to give the sameresult, namely a PrPres purification yield of between 80 and 100%; and

(4) the solubilization of said residue in a buffer C comprising at leastone surfactant, as defined in step (1), at a concentration of between0.1% and 5%, preferably of between 0.25% and 1%, based on the volume ofbuffer C (w/v), and/or at least one chaotropic agent at a concentrationof between 0.1 M and 8 M, at a temperature between room temperature and100° C., preferably equal to or greater than 80° C.; under suchtemperature conditions, the above-mentioned surfactants, preferablyionic surfactants, and/or the chaotropic agents solubilize the PrPres.

Said steps (1) and (2) can be carried out simultaneously orsuccessively; they are preferably carried out successively.

In one advantageous mode of carrying out said method, if the biologicalsample is a tissue or an organ, it is homogenized prior to step (1), forexample by mechanical grinding in a homogenization buffer consisting ofa neutral buffer such as water, or an isotonic buffer such as 5%glucose.

In another advantageous mode of carrying out said method, thetemperature used in step (1) is between room temperature and 50° C.; itis preferably 37° C.

Buffer A preferably comprises a surfactant selected from the groupconsisting of:

anionic surfactants such as SDS (sodium dodecylsulfate), sarkosyl(lauroylsarcosine), sodium cholate, sodium deoxycholate or sodiumtaurocholate;

zwitterionic surfactants such as SB 3-10 (decyl sulfobetaine), SB 3-12(dodecyl sulfobetaine), SB 3-14, SB 3-16 (hexadecyl sulfobetaine), CHAPSor deoxyCHAPS;

non-ionic surfactants such as C12E8 (dodecyl octaethylene glycol),Triton X100, Triton X114, Tween 20, Tween 80, MEGA 9(nonanoylmethylglucamine), octylglucoside, LDAO (dodecyldirethylamineoxide) or NP40; or

surfactant mixtures such as a mixture of an ionic surfactant and anon-ionic surfactant, especially the mixture SDS/Tween 80 or the mixturesarkosyl/Triton X100, a mixture of two ionic surfactants, such as themixture SDS/deoxycholate, or a mixture of an ionic surfactant and awitterionic surfactant.

In another advantageous mode of carrying out said method, buffer B ispreferably selected from C₃-C₆ alcohols and alcohol mixtures with a meantheoretical dielectric constant of between 10 and 25. The followingalcohols or alcohol mixtures are particularly preferred: butan-1-ol,butan-2-ol, 2-methylpropan-1-ol, isopropanol, isopropanol+pentanol,ethanol+hexanol, butanol+pentanol, etc.

In terms of the present invention, dielectric constant is understood asmeaning the static dielectric constant ε, measured in static orrelatively low frequency fields; it corresponds to the ratio of theelectric displacement D to the electric field strength E when anelectric field is applied to the solution at a temperature of between293.15 and 298.15 K.

The dielectric constant of liquids, as defined above, is described moreparticularly in CRC Handbook of Chemistry and Physics (ed. David R.Lide, 75 th edition, 1994, CRC Press).

For a solvent mixture, mean theoretical dielectric constant isunderstood as the mean of the dielectric constants of each solvent,weighted by its proportion in the mixture.

Surprisingly the addition of buffer B in step (2) makes it possible toobtain purification yields in excess of 90% under low speedcentrifugation conditions; it affords a significant reduction in theamount of final residue, while at the same time maintaining a highyield; advantageously the amount of final residue is preferably lessthan 10% of the initial weight of biological sample so as to be able toutilize it effectively in an immunoassay, whereas if only buffer A isadded, the resulting conditions are those of the prior art, whichnecessitate an ultracentrifugation in order to obtain sufficient yieldsof PrPres for the purposes of detection.

It may be noted that purification yields in excess of 80% can also beobtained by varying the centrifugation time and speed: 2 to 10 minutesat a speed below 20,000 g or a period of time reduced in proportion tothe increase in the number of g.

Preferably the ratio of the yield of PrPres in the solid phase to theamount of residue recovered after centrifigation of the suspension S2 isgreater than 10 when the initial sample corresponds to 100 mg of brain.

As a further preference, buffer C used in step (4) comprises achaotropic agent which is selected especially from the group consistingof urea and guanidine or a mixture thereof; it is also possible to useany other chaotropic agent.

The urea is preferably at a concentration of between 0.25 and 8 M andthe guanidine is preferably at a concentration of between 0.1 and 6 M.

If buffer C is a mixture of at least one surfactant and at least onechaotropic agent, it is preferably selected from the group consisting ofthe following mixtures: a mixture of SDS and urea, a mixture of sarkosyland urea, a mixture of deoxycholate and urea, a mixture of sarkosyl andguanidine or a mixture of sarkosyl, guanidine and urea Preferably, inthe mixture of SDS and urea, the SDS is at a concentration of 0.25-1%and the urea is at a concentration of 0.25-6 M; in the mixture ofsarkosyl and urea, the sarkosyl is at a concentration of between 0.25and 1% and the urea is at a concentration of between 0.25 and 8 M; inthe mixture of sarkosyl and guanidine, the sarkosyl is at aconcentration of between 0.25 and 1% and the guanidine is at aconcentration of between 0.5 M and 3 M, and in the mixture of sarkosyl,guanidine and urea, the sarkosyl is at a concentration of between 0.25and 1%, the guanidine is at a concentration of between 0.5 M and 3 M andthe urea is at a concentration of between 2 and 6 M.

Laemmli's buffer (4% SDS, 0.1 M Tris-HCl pH 8, 5% sucrose and 2%β-mercaptoethanol) can also be used, especially for western blotting.

The present invention further relates to a method of detecting PrPres ina biological sample, characterized in that it comprises:

treating said sample as defined above,

diluting the sample obtained, if necessary, and

detecting the PrPres by any appropriate analytical method, such as animmunological method (ELISA, western blotting), which produces aspecific signal.

The above-mentioned dilution step makes it possible to neutralize bufferC to enable detection of the PrPres by an ELISA method; it is effectedfor example with a buffer comprising albumin to give a final albuminconcentration of between 2 and 10% (w/v), or with a buffer based on 1%deoxycholate, for example.

A biological sample treated in this way contains an effectiveconcentration of PrPres, so the latter can be detected directly in saidsample by any analytical method, especially an immunological method.

As a variant, the present invention relates to a method of purifyingPrPres from a biological sample, characterized in that it comprisesessentially:

(1) the incubation, for 30 seconds to 2 hours, preferably for 30 secondsto 10 minutes, at a temperature below 80° C., of said biological samplewith a buffer A comprising at least one surfactant in an amount ofbetween a quarter and four times, preferably of between a quarter andone and a half times, the weight of the biological sample, andoptionally prior, subsequent or simultaneous incubation with a protease,to form an opalescent to turbid micellar or lamellar suspension S1;according to the invention, the protease is in fact added either before,after or simultaneously with the surfactant;

(2) the addition, to said micellar or lamellar suspension S1 obtained in(1), of a buffer B in an amount suitable for creating a phaseseparation, said buffer B consisting of a solvent or solvent mixturewhich does not solubilize the PrPres and has a dielectric constant ofbetween 10 and 25;

(3) the centrifigation of the suspension obtained in step (2); saidcentrifugation is carried out for example for 2 to 10 minutes at a speedbelow 20,000 g, preferably at a speed of between 3500 g and 17,500 g;the PrPres ends up at the interface;

(4) the recovery of the film present at the interface;

(5) the resolubilization of said film with a buffer A without theaddition of protease;

(6) the centrifugation of the suspension obtained in step (5) for 2 to10 minutes at a speed below 20,000 g, preferably at a speed of between3500 g and 17,500 g; the PrPres ends up in the centrifugation residuewith a PrPres purification yield surprisingly of between 70 and 100%;advantageously the centrifugation time and speed can be adapted to givethe same result, namely a PrPres purification yield of between 70 and100%; and

(7) the solubilization of said residue in a buffer C comprising at leastone surfactant, as defined in step (1), at a concentration of between0.1% and 5%, preferably of between 0.25% and 1%, based on the volume ofbuffer C (w/v), and/or at least one chaotropic agent at a concentrationof between 0.1 M and 8 M, at a temperature between room temperature and100° C., preferably equal to or greater than 80° C.; under suchtemperature conditions, the above-mentioned surfactants, preferablyionic surfactants, and/or the chaotropic agents solubilize the PrPres.

The amounts of buffer B to be added to give a microemulsion, amicro-suspension or phase separation are established with the aid of arange of buffers B, as illustrated for butan-1-ol in FIG. 1; they canvary as a function of buffer A and the constituents selected for bufferB.

As a variant, the solubilization step (step (4) of the first method orstep (7) of the second method) in said buffer C comprises heating at atemperature equal to or greater than 80° C. for 5 to 10 minutes,followed by centrifugation, preferably for 2 to 10 minutes, at a speedbelow 20,000 g, preferably at a speed of between 3500 g and 17,500 g; inthis case the PrPres is resolubilized and ends up in the supernatant;under such conditions, the sample obtained is particularly suitable forassaying the PrPres by an ELISA method.

The present invention further relates to a kit for treating a biologicalsample, characterized in that it comprises, in addition to a buffer forhomogenizing said biological sample, appropriate amounts of buffer A,buffer B and buffer C, as defined above.

The present invention further relates to a PrPres detection kit,characterized in that it comprises appropriate amounts of a buffer forhomogenizing the biological sample in which the PrPres is to bedetected, appropriate amounts of buffer A, buffer B and buffer C, asdefined above, and at least one appropriate anti-PrPres antibody.

Apart from the foregoing provisions, the invention also includes otherprovisions which will become apparent from the following descriptionreferring to Examples of how to carry out the method forming the subjectof the present invention, and to the attached drawings, in which:

FIG. 1 illustrates the influence of the amount of buffer B (butanol) onthe PrPres purification yield (in %) and the amount of residue obtainedafter centrifugation;

FIG. 2 illustrates the influence of different buffers B on the PrPrespurification yield (in %) and the amount of residue obtained aftercentrifugation;

FIG. 3 illustrates the role of the mean theoretical dielectric constantas defined above, in comparing different alcohol mixtures used as buffetB;

FIG. 4 illustrates an example of the detection of PrPres by westernblotting;

FIG. 5 illustrates the sensitivity and yield of the ELISA on differentdilutions of treated samples according to the invention;

FIG. 6 illustrates an ELISA performed directly on homogenates; and

FIG. 7 illustrates a comparison of different buffers C.

It must be clearly understood, however, that these Examples are givensolely as an illustration of the subject of the invention, without inany way implying a limitation.

EXAMPLE 1 Treatment of a Sample of Bovine Brain for Assaying PrPres inthe Field: Selection of the Appropriate Amount of Buffer B

A 500 mg sample of bovine brain is ground and homogenized to aconcentration of 25% (w/v) in 5% glucose solution.

To carry out the homogenization, the brain sample (500 mg) and 1 ml ofglucose are introduced into tubes containing ceramic beads, withagitation. The supernatant (about 1.5 ml) is recovered; the beads arerinsed in suspension in 500 μl of glucose and agitated; the supernatantobtained is recovered and mixed with the previous supernatant (2 ml).

400 μl of homogenate as obtained above (equivalent to 100 mg of brain)are incubated with 400 μl of buffer A comprising a mixture of equalparts of 25% (w/v) SDS and 25% (v/v) Tween 80 in a ratio of 1/1 (v/v)and proteinase K (0.1 mg/ml buffer A, i.e. 0.4 mg/g tissue) for 10minutes (2 times 5 min) at 37° C. (step (1)). As a variant, buffer Aadvantageously comprises 10% sarkosyl and 10% Triton X100; the latterbuffer A, which does not comprise SDS, is more advantageous because itdoes not cause any perturbation in the detection of the PrPres byspecific antibodies using a method of the ELISA type.

0 to 1000 μl of butanol (buffer B) are added (step (2)).

FIG. 1 shows the PrPres purification yield (%), quantified by westernblotting, and the amount of residue (in mg) as a function of the amountof buffer B added.

This Figure shows that between 10 and 53% of butanol the PrPres is keptin suspension, and that between 30 and 50% a purification yield of theorder of 100% and a residue of less than 10 mg are obtained.

After centrifugation at 3800 g (4000 rpm, JOUAN centrifuge) for 10minutes (step (3)), the supernatant is discarded and the residueobtained, which contains the PrPres, is dissolved in 80-100 μl of abuffer C (step (4)) comprising:

either 0.5% SDS and 0.5 M urea,

or Laemmli's buffer,

or 0.5% sarkosyl and 6 M urea, for 5 minutes at 100° C.

The PrPres is dissolved by this treatment; the sample is ready forimmediate use in an assay of the immunological type, such as ELISA orwestern blotting.

To perform an ELISA, the residue is preferably dissolved in a buffer Ccomprising sarkosyl (0.25-1%) and urea (0.25-8 M) or comprising SDS(0.25-1%) and urea (0.25-1 M); after heating, the sample obtained willpreferably be diluted (to ¼ or ½) with a buffer containing albumin togive a final albumin concentration of between 2 and 10% (w/v), or with abuffer containing 1% deoxycholate.

As a variant, the diluted sample is heated at 100° C. for 5-10 minutesand then centrifuged for 2 to 10 minutes at a speed below 20,000 g,preferably at a speed of between 3500 g and 17,500 g; the supernatant isdiluted to between ¼ and ½ with an ELISA buffer.

In the present case, the PrPres is quantified by western blotting, beingmixed with an equal volume of Laemmli's buffer; the diluted sample isthen deposited on gel for detection by western blotting; the amounts ofPrPres detected are compared with a linear range of dilutions of PrPrespurified under the same conditions as above from one and the samehomogenate of bovine brain affected by BSE, at the terminal stage of thedisease (positive control).

The samples treated by the method according to the invention are devoidof background and afford a reliable, specific and quantitative assay ofthe PrPres.

The method according to the invention allows a significant increase inthe PrPres purification yield:

In fact, if only buffer A is added, the yield of PrPres is of the orderof only 40%, so a loss of the order of 60% of the PrPres is observed,due especially to its partition between the solid phase and the liquidphase; moreover, there is a substantial amount of residue. This explainswhy the protocols described in the prior art use sarkosyl, which givesrise to smaller residues but necessitates cumbersomeultracentrifugations in order to obtain a sufficient yield.

Step (2) makes it possible to increase the yield of PrPres in the solidphase: a yield of the order of 80-100% is obtained in the solid phase ofthe, suspension S2 and the amount of residue is reduced at the sametime, as explained above.

EXAMPLE 2 Treatment of a Sample of Bovine Brain for Assaying PrPres inthe Field: Variant of Step (1) and Step (2)

The homogenization step is identical to that described in Example 1.

The 2 ml of homogenate obtained from the 500 mg of bovine brain are thenincubated with 2 ml of buffer A under the same conditions as thosedescribed in Example 1 (step (1)).

3 ml of buffer B are added under the same conditions as those describedin Example 1 (step (2)).

The remainder of the method is identical to that of Example 1.

EXAMPLE 3 Treatment of a Sample of Bovine Brain for Assaying PrPres inthe Field: Variant of the Homogenization Step

A 250 mg sample of bovine brain is ground and homogenized to aconcentration of 25% (w/v) in 5% glucose solution.

To carry out the homogenization, the sample of brain (250 mg) and 750 μlof glucose are introduced into tubes containing ceramic beads, withagitation for 40 seconds (RIBOLYSER-HYBAID apparatus); 400 μl ofsupernatant arc withdrawn and the remainder of the procedure is as inExample 1.

EXAMPLE 4 Comparison of the Influence of Different Buffers B on theRatio of the PrPres Purification Yield to the Amount of Residue

The sample is treated under the same conditions as those described inExample 1 except that the amount of buffer B is 600 μl.

FIG. 2 illustrates the ratios obtained by western blotting withdifferent buffers B: pentanol, butanol, isopropanol/pentanol mixture,ethanol/hexanol mixture, isopropanol and ethanol; the mixtures were madeup by volume.

EXAMPLE 5 Comparison of the Dielectric Constants of Different Mixturesand Their Influence on the PrPres Purification Yield and the Amount ofResidue

The method is carried out under the conditions described in Example 4.

FIG. 3 illustrates the results obtained: in the case where buffer B is amixture of alcohols, the volume of each alcohol is calculated as afunction of its dielectric constant and the desired theoreticaldielectric constant of the mixture (for example 17) and based on a totalvolume of alcohol of 600 μl. For example, the following formula isobtained for the hexanol/ethanol mixture:

13.y+25(1−y)=17

y being the percentage of hexanol, 13 being the dielectric constant ofhexanol and 25 being the dielectric constant of ethanol.

For a mean theoretical dielectric constant of 15 or below, phaseseparation is observed.

EXAMPLE 6 Detection of PrPres by Western Blotting

*Protocol:

1) Incubation at 37° C., for 2 times 5 min. of 400 mg of 25%(weight/volume) bovine brain homogenate and 400 μl of buffer Acomprising 400 μl of a mixture of equal parts (v/v) of 25% (w/v) SDS and25% (v/v) Tween 80 (50/50) and proteinase K (PK) at a concentration of0.1 mg/ml buffer A.

2) 600 μl of buffer B (or 1000 μl of buffer B for the samples of lanes 7and 8), consisting of butan-1-ol, are added.

3) The mixture is centrifuged at 15,000 rpm for 5 min (about 17,000 g).

4) The centrifugation residue is taken up in 100 pi of Laemmli's buffercontaining 4% SDS, and heated at 100° C. for 5 min.

*Western Blotting:

The samples obtained are used to perform SDS-PAGE and transferred to anitrocellulose membrane under the conditions described by Towbin et al.(Proc. Natl. Acad. Sci. USA, 1979, 76, 4350-4354) or by C. I. Lasmézaset al. (J. Gen. Virol., 1996, op. cit.).

Before being deposited on the electrophoresis gel, the samples werediluted to 1/20 in a negative control produced under the same conditionsas those described in Example 1 from a healthy bovine homogenate,because of the magnitude of the signals (12% polyacrylamide gel loadedwith the equivalent of 10 mg (10 μl) of brain, corresponding to 9.5 mgof healthy bovine brain and 0.5 mg of infected bovine brain).

Immunodetection of the PrPres was effected with the antiserum JB007 (R.Demaimay et al., J. Virol., 1997, 71, 12, 9685-9689) (1/5000) andanti-rabbit goat Ig conjugated with peroxidase (1/2500). Theimmunoreactivity is revealed by chemiluminescence (ECL, Amersham),quantified and visualized on autoradio-graphic films, as illustrated inFIG. 4.

FIG. 4 illustrates the results obtained and corresponds to thepropanol/hexanol curve of FIG. 3A.

In FIG. 4, lanes 1 to 6 correspond to samples treated using differentpropanol/hexanol mixtures as buffer B, leading to different meantheoretical dielectric constants; lanes 8 and 9 correspond to biologicalsamples subjected to a procedure using 1000 μl of butanol as buffer B(53% on the abscissa of FIG. 1): it is observed in this case that allthe PrPres ends up at the interface; lanes 9 and 10 correspond tobiological samples subjected to a procedure using 600 μl of butanol asbuffer B (43% on the abscissa of FIG. 1): it is observed in this casethat all the PrPres ends up in the residue (lane 10), whereas no signalis observed in a negative control treated under the same conditions(lane 9).

EXAMPLE 7 ELISA of PrPres from a Sample Obtained According to theInvention

A sample is prepared under the following conditions:

A 400 mg sample of bovine brain is ground and homogenized to aconcentration of 20% in 5% glucose solution (1.6 ml) under the sameconditions as those described in Example 1.

500 μl of the homogenate obtained arc incubated at 37° C. for 10 min (2times 5 min with intermediate agitation) with 500 μl of buffer Acomprising 10% sarkosyl and 10% Triton X100, together with proteinase K(80 μg/ml).

500 μl of buffer B (butanol) are added.

The mixture is centrifuged for 5 min at 15,000 rpm with a rotor capableof producing 17,608 g; the same results are obtained with acentrifugation time of 4 min at 20,627 g.

The supernatant is discarded and the residue obtained, which containsPrPres, is dissolved for 5 min at 10° C. in 100 μl of buffer Ccomprising 6 M urea and 0.5% sarkosyl.

The mixture is centrifuged for 5 min at 15,000 rpm with a rotor capableof producing 17,608 g; the same results are obtained with acentrifugation time of 4 min at 20,627 g. The supernatant is diluted to1/3 in EIA buffer (phosphate buffer with 0.5% deoxycholate).

To perform the ELISA, the sample obtained is deposited at a rate of 100μl per well, in duplicate, on a plate covered with a first anti-PrPantibody (8G8, ref. Kraseman et al., Molecular Medicine, 1996); this isfollowed by incubation, washing and developing with a second anti-PrPantibody (12F10, ref. Kraseman et al., 1996) coupled withacetylcholinesterase (ref. Grassi et al., J. Immunol. Methods, 1989).After incubation with the substrate for acetylcholinesterase, theresults are read at 415 nm 15 min or 30 min after addition of thesubstrate (Ellmann's reagent).

The results shown in FIG. 5 relate to a 20% (weight/volume) homogenateof bovine brain clinically affected by BSE, diluted to 1/10, 1/100,1/1000 or 1/10,000 in a 20% (weight/volume) homogenate of healthy bovinebrain.

The samples are then treated under the conditions described above andtested by EIA with 8G8 as the capturing antibody and 12F10 as thedetecting antibody.

The samples are tested diluted in the EIA depot buffer (dilutions to1/3, then by factors of 3).

FIG. 5 illustrates the results obtained (optical density of the signalas a function of the final dilution of the homogenate, i.e. dilution inthe negative homogenate×dilution in the EIA buffer): the semilogarithmiccurve has the sigmoid shape conventionally observed in all assays of theEIA type. Furthermore, it is pointed out that there is a goodsuperposition of the curves obtained from the homogenates diluted to1/10, 1/100 and 1/1000. Finally, the sensitivity level of the test makesit possible to detect this positive brain diluted to 1/10,000.

In FIG. 6, another homogenate diluted to 1/100 was tested by the EIAtechnique, either after treatment as described above with a PKconcentration of 400 ng/mg, or after direct treatment of the homogenateswith different PK concentrations. It is found that direct treatment ofthe homogenates with PK, without purification, gives either high signalsof the negative controls for low PK concentrations (incompletedestruction of PrPc), or low signals for the positive cows with high PKconcentrations. This Example therefore emphasizes that it is essentialto treat the homogenates in the manner described above if the PrPres isto detected correctly.

EXAMPLE 8 Comparison of Different Buffers C

The sample treatment protocol used is that of Example 7.

FIG. 7 illustrates the results obtained.

Guanidine seems to be advantageous and the urealguanidine combinationappears to be the most valuable (in the knowledge that urea is bettertolerated by ELISA).

As is apparent from the foregoing description, the invention is in noway limited to those modes of execution, embodiments and modes ofapplication which have now been described more explicitly, on thecontrary, it encompasses all the variants thereof which may occur tothose skilled in the art, without deviating from the framework or thescope of the present invention.

What is claimed is:
 1. A method of purifying abnormal form of prionprotein (PrPres) from a biological sample, comprising therefor: (1) theincubation, for 30 seconds to 2 hours, at a temperature below 80° C., ofsaid biological sample with a buffer A comprising at least onesurfactant in an amount of between a quarter and four times the weightof the biological sample, and optionally prior, subsequent orsimultaneous incubation with a protease, to form a suspension S1; (2)the addition, to said suspension S1 obtained in (1), of a buffer B in anamount suitable for clarifying said suspension by forming amicroemulsion or a microsuspension, said buffer B consisting of asolvent or solvent mixture which does not solubilize the PrPres and hasa dielectric constant of between 10 and 25, to form a suspension S2; (3)the centrifugation of the suspension S2 to form a residue; and (4) thesolubilization of said residue in a buffer C comprising at least onecompound selected from the group consisting of: (i) one surfactant, asdefined in step (1), at a concentration of between 0.1% and 5%, based onthe volume of buffer C (w/v), (ii) one chaotropic agent at aconcentration of between 0.1 M and 8 M, and (iii) a mixture of compound(i) and compound (ii), at a temperature between room temperature and100° C., to form a solution.
 2. The method according to claim 1, whereinin step 1 the incubation time is between 30 seconds and 10 minutes. 3.The method according to claim 1, wherein buffer A comprises a surfactantselected from the group consisting of: anionic surfactants such as SDS(sodium dodecylsulfate), sarkosyl (lauroylsarcosine), sodium cholate,sodium deoxycholate or sodium taurocholate; zwitterionic surfactantssuch as SB 3-10 (decyl sulfobetaine), SB 3-12 (dodecyl sulfobetaine), SB3-14, SB 3-16 (hexadecyl sulfobetaine), CHAPS or deoxyCHAPS; non-ionicsurfactants such as C12E8 (dodecyl octaethylene glycol), Triton X100,Triton X114, Tween 20, Tween 80, MEGA 9 (nonanoylmethylglucamine),octylglucoside, LDAO (dodecyldimethylamine oxide) or NP40; andsurfactant mixtures such as a mixture of an ionic surfactant and anon-ionic surfactant, a mixture of two ionic surfactants or a mixture ofan ionic surfactant and a zwitterionic surfactant.
 4. The methodaccording to claim 1, wherein in step (1), the amount of surfactantpresent in buffer A is between a quarter and one and a half times theweight of the biological sample.
 5. The method according to claim 1,wherein buffer B is selected from the group consisting of C₃-C₆ alcoholsand alcohol mixtures with a mean theoretical dielectric constant ofbetween 10 and
 25. 6. The method according to claim 1, wherein thefollowing alcohols or alcohol mixtures are selected from the groupconsisting of butan-1-ol, butan-2-ol, 2-methylpropan-1-ol, isopropanol,isopropanol+pentanol, ethanol+hexanol and butanol+pentanol.
 7. Themethod according to claim 1, wherein buffer C comprises a chaotropicagent which is selected from the group consisting of urea, guanidine,and mixtures of urea and guanidine.
 8. The method according to claim 1,wherein the solubilization step comprises heating at a temperature equalto or greater than 80° C. for 5 to 10 minutes, followed bycentrifugation.
 9. The method of claim 1, wherein the concentration ofthe surfactant in buffer C is between 0.25% and 1% and the temperatureof the solubilization step is between 80° C. and 100° C.
 10. A method ofclaim 1, further comprising the step of homogenizing the biologicalsample before it is suspended in Buffer A.
 11. The method according toclaim 1, wherein buffer C comprises a surfactant selected from the groupconsisting of: anionic surfactants selected from the group consisting ofSDS (sodium dodecylsulfate), sarkosyl (lauroylsarcosine), sodiumcholate, sodium deoxycholate or sodium taurocholate; zwitterionicsurfactants selected from the group consisting of SB 3-10 (decylsulfobetaine), SB 3-12 (dodecyl sulfobetaine), SB 3-14, SB 3-16(hexadecyl sulfobetaine), CHAPS or deoxyCHAPS; non-ionic surfactantsselected from the group consisting of C12E8 (dodecyl octaethyleneglycol), Triton X100, Triton X114, Tween 20, Tween 80, MEGA 9(nonanoylmethylglucamine), octylglucoside, LDAO (dodecyldimethylamineoxide) or NP40; and surfactant mixtures selected from the groupconsisting of a mixture of an ionic surfactant and a non-ionicsurfactant, a mixture of two ionic surfactants or a mixture of an ionicsurfactant and a zwitterionic surfactant.
 12. A method of claim 1,further comprising: (1) the addition at step 2, to said suspension S1,of buffer B in an amount suitable for creating a phase separation; (2)following centrifugation of suspension S2, recovery of a film present atthe interface; (3) resolubilization of the film with buffer A withoutthe addition of protease; (4) centrifugation of a suspension obtained instep (3) to produce a residue soluble in buffer C.
 13. The method ofclaim 12, wherein the concentration of the surfactant in buffer C isbetween 0.25% and 1% and the temperature of the solubilization step isbetween 80° C. and 100° C.
 14. The method according to claim 1, whereinthe temperature used in step (1) is between room temperature and 50° C.15. The method of claim 14, wherein the temperature used in step 1 isabout 37° C.
 16. The method according to claim 1, wherein thecentrifugation of step (3) is carried out for 2 to 10 minutes at a speedbelow 20,000 g.
 17. The method of claim 16, wherein the centrifugationspeed is between 3500 g and 17,500 g.
 18. A method of claim 1, furthercomprising the step of detecting the PrPres contained in the solutionobtained at the end of step 4 by reacting it with a labeled anti-PrPresantibody.
 19. A method of claim 18, wherein the solution containing thePrPres is diluted prior to the detection step.
 20. A kit for treating abiological sample for the purpose of purifying PrPres from saidbiological sample, comprising, in addition to a buffer for homogenizingsaid biological sample, appropriate amounts of (i) a buffer A, selectedfrom the group consisting of anionic surfactants selected from the groupconsisting of SDS (sodium dodecylsulfate), sarkosyl (lauroylsarcosine),sodium cholate, sodium deoxycholate and sodium taurocholate;zwitterionic surfactants selected from the group consisting of SB 3-10(decyl sulfobetaine), SB 3-12 (dodecyl sulfobetaine), SB 3-14, SB 3-16(hexadecyl sulfobetaine), CHAPS and deoxyCHAPS; non-ionic surfactantsselected from the group consisting of C12E8 (dodecyl octaethyleneglycol), Triton X100, Triton X114, Tween 20, Tween 80, MEGA 9(nonanoylmethylglucamine), octylglucoside, LDAO (dodecyldimethylamineoxide) and NP40; and surfactant mixtures selected from the groupconsisting of a mixture of an ionic surfactant and a non-ionicsurfactant, a mixture of two ionic surfactants and a mixture of an ionicsurfactant and a zwitterionic surfactant; (ii) a buffer B selected fromthe group consisting of C3-C6 alcohols and alcohol mixtures with a meantheoretical dielectric constant of between 10 and 25; (iii) a buffer Cwhich comprises a chaotropic agent which is selected from the groupconsisting of urea, guanidine, and mixtures of urea and guanidine; and(iv) an anti-PrPres antibody.